CN110101880A - One kind being based on 2PisoDGR2Radiopharmaceutical of polypeptide and preparation method thereof - Google Patents
One kind being based on 2PisoDGR2Radiopharmaceutical of polypeptide and preparation method thereof Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 50
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 39
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 37
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 37
- 239000000539 dimer Substances 0.000 claims abstract description 40
- 102000001189 Cyclic Peptides Human genes 0.000 claims abstract description 37
- 108010069514 Cyclic Peptides Proteins 0.000 claims abstract description 37
- 239000012217 radiopharmaceutical Substances 0.000 claims abstract description 24
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 20
- 239000000178 monomer Substances 0.000 claims abstract description 20
- 239000002738 chelating agent Substances 0.000 claims abstract description 15
- 239000003814 drug Substances 0.000 claims abstract description 8
- 229940079593 drug Drugs 0.000 claims abstract description 7
- 238000006471 dimerization reaction Methods 0.000 claims abstract description 6
- 239000004475 Arginine Substances 0.000 claims abstract description 5
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 43
- 238000000034 method Methods 0.000 claims description 32
- 238000006243 chemical reaction Methods 0.000 claims description 31
- 239000007788 liquid Substances 0.000 claims description 27
- WDLRUFUQRNWCPK-UHFFFAOYSA-N Tetraxetan Chemical compound OC(=O)CN1CCN(CC(O)=O)CCN(CC(O)=O)CCN(CC(O)=O)CC1 WDLRUFUQRNWCPK-UHFFFAOYSA-N 0.000 claims description 23
- 229940121896 radiopharmaceutical Drugs 0.000 claims description 23
- 230000002799 radiopharmaceutical effect Effects 0.000 claims description 23
- 239000000243 solution Substances 0.000 claims description 21
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims description 18
- JHALWMSZGCVVEM-UHFFFAOYSA-N 2-[4,7-bis(carboxymethyl)-1,4,7-triazonan-1-yl]acetic acid Chemical compound OC(=O)CN1CCN(CC(O)=O)CCN(CC(O)=O)CC1 JHALWMSZGCVVEM-UHFFFAOYSA-N 0.000 claims description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 16
- 238000010828 elution Methods 0.000 claims description 13
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims description 10
- SEQKRHFRPICQDD-UHFFFAOYSA-N N-tris(hydroxymethyl)methylglycine Chemical compound OCC(CO)(CO)[NH2+]CC([O-])=O SEQKRHFRPICQDD-UHFFFAOYSA-N 0.000 claims description 10
- 238000004108 freeze drying Methods 0.000 claims description 9
- 238000001816 cooling Methods 0.000 claims description 8
- KDYFGRWQOYBRFD-UHFFFAOYSA-N succinic acid Chemical compound OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 claims description 8
- CGIHPACLZJDCBQ-UHFFFAOYSA-N acibenzolar Chemical compound SC(=O)C1=CC=CC2=C1SN=N2 CGIHPACLZJDCBQ-UHFFFAOYSA-N 0.000 claims description 6
- 238000001914 filtration Methods 0.000 claims description 6
- 229910005267 GaCl3 Inorganic materials 0.000 claims description 5
- UZMAPBJVXOGOFT-UHFFFAOYSA-N Syringetin Natural products COC1=C(O)C(OC)=CC(C2=C(C(=O)C3=C(O)C=C(O)C=C3O2)O)=C1 UZMAPBJVXOGOFT-UHFFFAOYSA-N 0.000 claims description 5
- 239000007997 Tricine buffer Substances 0.000 claims description 5
- 230000008859 change Effects 0.000 claims description 5
- KCFYHBSOLOXZIF-UHFFFAOYSA-N dihydrochrysin Natural products COC1=C(O)C(OC)=CC(C2OC3=CC(O)=CC(O)=C3C(=O)C2)=C1 KCFYHBSOLOXZIF-UHFFFAOYSA-N 0.000 claims description 5
- ZDQYSKICYIVCPN-UHFFFAOYSA-L sodium succinate (anhydrous) Chemical compound [Na+].[Na+].[O-]C(=O)CCC([O-])=O ZDQYSKICYIVCPN-UHFFFAOYSA-L 0.000 claims description 5
- MYAJTCUQMQREFZ-UHFFFAOYSA-K tppts Chemical compound [Na+].[Na+].[Na+].[O-]S(=O)(=O)C1=CC=CC(P(C=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=C(C=CC=2)S([O-])(=O)=O)=C1 MYAJTCUQMQREFZ-UHFFFAOYSA-K 0.000 claims description 5
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 claims description 4
- 230000004913 activation Effects 0.000 claims description 4
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 4
- 229910052733 gallium Inorganic materials 0.000 claims description 4
- 235000013922 glutamic acid Nutrition 0.000 claims description 4
- 239000004220 glutamic acid Substances 0.000 claims description 4
- 239000001384 succinic acid Substances 0.000 claims description 4
- GYHNNYVSQQEPJS-UHFFFAOYSA-N Gallium Chemical compound [Ga] GYHNNYVSQQEPJS-UHFFFAOYSA-N 0.000 claims description 3
- 239000002253 acid Substances 0.000 claims description 3
- 239000007864 aqueous solution Substances 0.000 claims description 3
- 238000010438 heat treatment Methods 0.000 claims description 3
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims description 2
- 230000015572 biosynthetic process Effects 0.000 claims description 2
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- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 40
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 description 16
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 15
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- WSLDOOZREJYCGB-UHFFFAOYSA-N 1,2-Dichloroethane Chemical compound ClCCCl WSLDOOZREJYCGB-UHFFFAOYSA-N 0.000 description 2
- JGSARLDLIJGVTE-UHFFFAOYSA-N 3,3-dimethyl-7-oxo-6-[(2-phenylacetyl)amino]-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid Chemical compound O=C1N2C(C(O)=O)C(C)(C)SC2C1NC(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-UHFFFAOYSA-N 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
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- 208000026310 Breast neoplasm Diseases 0.000 description 2
- OAKJQQAXSVQMHS-UHFFFAOYSA-N Hydrazine Chemical compound NN OAKJQQAXSVQMHS-UHFFFAOYSA-N 0.000 description 2
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- NAQWICRLNQSPPW-UHFFFAOYSA-N 1,2,3,4-tetrachloronaphthalene Chemical compound C1=CC=CC2=C(Cl)C(Cl)=C(Cl)C(Cl)=C21 NAQWICRLNQSPPW-UHFFFAOYSA-N 0.000 description 1
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 1
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 description 1
- PEJJTUMTZBUFGS-UHFFFAOYSA-N C1(=CC=CC=C1)P.[Na] Chemical compound C1(=CC=CC=C1)P.[Na] PEJJTUMTZBUFGS-UHFFFAOYSA-N 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- 208000037845 Cutaneous squamous cell carcinoma Diseases 0.000 description 1
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 description 1
- 206010014759 Endometrial neoplasm Diseases 0.000 description 1
- 102100032817 Integrin alpha-5 Human genes 0.000 description 1
- 108010041014 Integrin alpha5 Proteins 0.000 description 1
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- 235000006508 Nelumbo nucifera Nutrition 0.000 description 1
- 235000006510 Nelumbo pentapetala Nutrition 0.000 description 1
- DFPAKSUCGFBDDF-UHFFFAOYSA-N Nicotinamide Chemical compound NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 description 1
- 108010077895 Sarcosine Proteins 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 208000000102 Squamous Cell Carcinoma of Head and Neck Diseases 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 102000019997 adhesion receptor Human genes 0.000 description 1
- 108010013985 adhesion receptor Proteins 0.000 description 1
- BJEPYKJPYRNKOW-UHFFFAOYSA-N alpha-hydroxysuccinic acid Natural products OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 description 1
- 150000001413 amino acids Chemical group 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000001588 bifunctional effect Effects 0.000 description 1
- OVYQSRKFHNKIBM-UHFFFAOYSA-N butanedioic acid Chemical compound OC(=O)CCC(O)=O.OC(=O)CCC(O)=O OVYQSRKFHNKIBM-UHFFFAOYSA-N 0.000 description 1
- 230000021164 cell adhesion Effects 0.000 description 1
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- 239000012295 chemical reaction liquid Substances 0.000 description 1
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- 238000012937 correction Methods 0.000 description 1
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- 239000012467 final product Substances 0.000 description 1
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- 150000002466 imines Chemical class 0.000 description 1
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- 238000001727 in vivo Methods 0.000 description 1
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- 230000005764 inhibitory process Effects 0.000 description 1
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- 238000010253 intravenous injection Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
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- 230000036210 malignancy Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 210000000214 mouth Anatomy 0.000 description 1
- 230000001537 neural effect Effects 0.000 description 1
- 229960003966 nicotinamide Drugs 0.000 description 1
- 235000005152 nicotinamide Nutrition 0.000 description 1
- 239000011570 nicotinamide Substances 0.000 description 1
- 208000010655 oral cavity squamous cell carcinoma Diseases 0.000 description 1
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- 238000010837 poor prognosis Methods 0.000 description 1
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- 238000004809 thin layer chromatography Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- SOBHUZYZLFQYFK-UHFFFAOYSA-K trisodium;hydroxy-[[phosphonatomethyl(phosphonomethyl)amino]methyl]phosphinate Chemical compound [Na+].[Na+].[Na+].OP(O)(=O)CN(CP(O)([O-])=O)CP([O-])([O-])=O SOBHUZYZLFQYFK-UHFFFAOYSA-K 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
- A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
- A61K51/04—Organic compounds
- A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
- A61K51/082—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins the peptide being a RGD-containing peptide
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Pharmacology & Pharmacy (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Physics & Mathematics (AREA)
- Optics & Photonics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Peptides Or Proteins (AREA)
- Medicinal Preparation (AREA)
Abstract
The invention discloses one kind to be based on 2PisoDGR2Radiopharmaceutical of polypeptide and preparation method thereof, including isoDGR cyclic peptide dimer and radionuclide, the isoDGR cyclic peptide dimer are to be connected with bridging agent PEG for two4Ring isoRGD polypeptide monomer dimerization and synthesize, the sequence of the ring isoRGD polypeptide monomer is different aspartic-glycine-arginine；The radionuclide marks the isoDGR cyclic peptide dimer by chelating agent.Drug of the present invention gathers tumor locus by the way that the targeting of isoDGR polypeptide is dense, more accurate to integrin a using the single photon tomography technology or Positron Emission Computed Tomography technology of nuclear medicinevb6With integrin a5b1Double positive and integrin avb6Or integrin a5b1The localization diagnosis of single positive tumor patient.
Description
Technical field
The present invention relates to radiopharmaceutical preparation field, specially a kind of radiopharmaceutical based on isoDGR polypeptide and
Preparation method.
Background technique
Integrin is a kind of cell adhesion receptor being made of α and β Liang Ge subunit, participates in the signal transduction of intraor extracellular
To regulate and control various important cell functions, such as adherency, polarity, differentiation, migration and cell division.Tumour is in occurrence and development
It is related to tumor vascular newborn and integrin expression in journey to change.Integrin alphavβ6And α5β1In embryo development procedure all
There is specific high expression, and plays an important role.Integrin alphavβ6And α5β1With the spy of low expression in the adult of health
Property, but there is the high of specificity to express during the occurrence and development of disease or tumour.Integrin alphavβ6In cancer of pancreas, breast cancer, lung
Up-regulation is expressed in cancer, oral cavity and cutaneous squamous cell carcinoma, colon cancer, gastric cancer and carcinoma of endometrium.Integrin alpha5β1In Colon and rectum
High expression in a variety of tumours such as cancer, breast cancer, oophoroma, lung cancer, gastric cancer and neural glioma.Integrin alphavβ6And α5β1
High expression be directed to the malignancy and poor prognosis of tumour, while be also exploitation tumor developer important target spot it
One.
Different aspartic-glycine-arginine (isoDGR) sequence is to be different from RGD (arginine-glycine-asparagus fern ammonia
Acid) sequence targeted integration element family new amino acid sequence.Existing research the result shows that, c (phg-isoDGRk) polypeptide
(referred to as isoDGR polypeptide) energy and integrin alpha5β1And αvβ6With the combination of higher affinity.Although isoDGR has well
Clinical landscapes, but isoDGR is applied to radiopharmaceutical class product and still has many deficiencies, at present about isoDGR polypeptide
The research of optimization and product still belong to blank.
Summary of the invention
The purpose of the present invention is to provide a kind of novel radiopharmaceutical based on isoDGR, compared to existing
IsoDGR can enhance the receptor-ligand affinity of drug to reach higher tumor uptake, so as to be used for integrin alphav
β6With integrin alpha5β1Double positive and integrin alphasvβ6Or integrin alpha5β1The localization diagnosis of single positive tumor patient.
The purpose of the present invention is be achieved through the following technical solutions:
One kind being based on 2PisoDGR2The radiopharmaceutical of polypeptide, including isoDGR cyclic peptide dimer and radionuclide, institute
Stating isoDGR cyclic peptide dimer is to be connected with bridging agent PEG for two4Ring isoRGD polypeptide monomer dimerization and synthesize,
Structure is as shown in Figure 1, the sequence of the ring isoRGD polypeptide monomer is different aspartic-glycine-arginine；The radioactivity
Nucleic marks the isoDGR cyclic peptide dimer by chelating agent.
Further, the chelating agent is HYNIC, any one in DOTA, NOTA；The radionuclide is68Ga
Or99mTc。
A kind of scheme advanced optimized, the radionuclide are68Ga, the chelating agent are DOTA or NOTA.
Another scheme advanced optimized is also connected with medicine between the isoDGR cyclic peptide dimer and the chelating agent
For dynamics decorating molecule bridging agent, the pharmacokinetics decorating molecule bridging agent is PEGn, wherein n=1~9.
Further, the radionuclide is99mTc, the chelating agent are HYNIC,
Further, the pharmacokinetics decorating molecule bridging agent is PEG4。
Further, the radiopharmaceutical is colourless transparent liquid injection.
The 2PisoDGR2The preparation method of polypeptide radiopharmaceutical, wherein the preparation side of isoDGR cyclic peptide dimer
Method are as follows:
PEG will be connected with4The ring isoDGR polypeptide monomer of bridging agent is dissolved in DMF；Boc-protected N- hydroxy succinic acid is added
The glutamic acid Acibenzolar of imines activation；PH is adjusted with DIEA to adjust to 8.0-9.0；Reaction solution is isolated and purified through HPLC method, is received
Collect the fraction of target product, merge collection liquid and is lyophilized；TFA is added in the product of acquisition, outstanding to boil off except TFA, residue pure water
Dissolution, freeze-drying obtains the isoDGR cyclic peptide dimer after filtering.
A kind of further preparation method is that preparation method includes following preparation step:
a.HYNIC-PEGnThe preparation of-NHS:
By amino-PEGnOrganic acid is dissolved in pure water, adjusts pH to 8.0-9.0；HYNIC-NHS is dissolved in DMF, is added
Enter to regulate the amino-PEG of pH valuenAqueous solutions of organic acids；It is stirred at room temperature；Reaction solution is isolated and purified through HPLC method, collects mesh
The fraction of product is marked, collection liquid is merged and is lyophilized, HYNIC-PEG is obtainedn-COOH；By HYNIC-PEGn- COOH is dissolved in DMF,
EDC and NHS is added, is stirred at room temperature；Reaction solution is isolated and purified through HPLC method, collects the fraction of target product, merges collection liquid
And it is lyophilized and obtains HYNIC-PEGn-NHS；
b.HYNIC-PEGn-E[PEG4-c(phg-isoDGRk)]2Preparation:
The isoDGR cyclic peptide dimer is expressed as E [PEG4-c(phg-isoDGRk)]2, by isoDGR cyclic peptide dimer and
HYNIC-PEGn- NHS is dissolved in DMF, is adjusted pH to 8.0-9.0 with DIEA, is stirred at room temperature；Product separates pure through HPLC method
Change, collect target fraction, merges collection liquid and freeze-drying obtains HYNIC-PEGn-E[PEG4-c(phg-isoDGRk)]2；
c.99mTc-HYNIC-PEGn-E[PEG4-c(phg-isoDGRk)]2Preparation:
Configuration contains TPPTS, tricine, disodium succinate, succinic acid and HYNIC-PEGn-E[PEG4-c(phg-
isoDGRk)]2Mixed liquor, be added Na99mTcO4Solution, 100 DEG C heating in water bath for reaction 15-20 minutes, to room after reaction
Temperature is cooling, is made99mTc-HYNIC-PEGn-E[PEG4-c(phg-isoDGRk)]2, as it is based on 2PisoDGR2The radiation of polypeptide
Property drug.
Further another kind preparation method is that preparation method includes following preparation step:
a.NOTA(DOTA)-E[PEG4-c(phg-isoDGRk)]2Preparation:
The isoDGR cyclic peptide dimer is expressed as E [PEG4-c(phg-isoDGRk)]2, by the isoDGR cyclic peptide dimerization
Body and NOTA-NHS or DOTA-NHS are dissolved in 1.0mL DMF, are adjusted pH with DIEA and are adjusted to 8.0-9.0, are stirred at room temperature；Reaction
Liquid is isolated and purified through HPLC method, collects the fraction of target product, is merged collection liquid and is lyophilized, obtains product NOTA (DOTA)-E
[PEG4-c(phg-isoDGRk)]2；
b.68Ga-NOTA(DOTA)-E[PEG4-c(phg-isoDGRk)]2Preparation:
From germanium-fresh elution of gallium generator68GaCl3, use NH4OAc adjusts pH to 3.0~4.0, and NOTA (DOTA)-E is added
[PEG4-c(phg-isoDGRk)]2, 99 DEG C are heated 19~21 minutes, and it is cooling to room temperature after reaction, it is made68Ga-NOTA
(DOTA)-E[PEG4-c(phg-isoDGRk)]2, as it is based on 2PisoDGR2The radiopharmaceutical of polypeptide.
The beneficial effects of the present invention are:
The present invention is connected with pharmacokinetics bridging agent PEG for two4IsoRGD polypeptide monomer dimerization and synthesize
IsoDGR cyclic peptide dimer.IsoDGR cyclic peptide dimer has the internal pharmacokinetics of higher affinity and improvement compared with monomer
Matter.The drug is by chelating agent by radioisotope labeling to isoDGR cyclic peptide dimer molecule, and labeled drug is logical in vivo
Cross isoDGR polypeptide targeting it is dense gather tumor locus, using nuclear medicine single photon tomography (SPECT) technology or
Positron Emission Computed Tomography technology (PET), it is more accurate to integrin alphavβ6With integrin alpha5β1It is double it is positive, with
And integrin alphavβ6Or integrin alpha5β1The localization diagnosis of single positive tumor patient.
Detailed description of the invention
Fig. 1 is the structure chart of isoDGR cyclic peptide dimer；
Fig. 2 is that isoDGR cyclic peptide dimer is connected with bridging agent (PEGn, n=1~9 are indicated with PKM) and bifunctional chelating agent
The structure chart of (HYNIC, NOTA, DOTA etc.)；
Fig. 3 is the isoDGR cyclic peptide dimer and isoDGR monomer pair of various concentration125I-c (y-isoDGR-k) and U87-
The drug fixed double chamber bed of the competitive binding curve (A) of MG cell combination and the isoDGR dimer of mtc labeled and isoDGR monomer
Curve (B)；
Fig. 4 is HP-2PisoDGR2(HYNIC-PEG4-E[PEG4-c(phg-isoDGRk)]2) chemical structural formula (A),99mTc-HP-2PisoDGR2(99mTc-HYNIC-PEG4-E[PEG4-c(phg-isoDGRk)]2) mark structure schematic diagram (B) with
Radioactivity HPLC (high performance liquid chromatography) spectrogram (C), and in integrin alpha5β1Positive U87MG human glioma tumor bearing nude mice note
It penetrates99mTc-HP-2PisoDGR20.5, the 1 and 2h and toy SPECT/CT with the excessive cold closed control group 0.5h of peptide afterwards
Imaging figure (D), arrow represent tumour (posterior view, rearview)；
Fig. 5 is intravenous injection99mTc-HP-2PisoDGR21 hour SPECT/CT in the spontaneous pancreatic cancer models of KPC afterwards
After imaging figure (A), and injection after 2 hours SPECT/CT in vitro 3D imaging figure (B), arrow locations indicate spontaneous pancreas in situ
Gland cancer (posterior view, rearview)；
Fig. 6 is NOTA-2PisoDGR2(NOTA-E[PEG4-c(phg-isoDGRk)]2) chemical structural formula (A),68Ga-
2PisoDGR2(68Ga-NOTA-E[PEG4-c(phg-isoDGRk)]2) mark structure schematic diagram (B) and ITLC (radio thin layer
Chromatography) spectrogram (C), and in integrin alpha5β1Positive U87MG human glioma tumor bearing nude mice injection68Ga-2PisoDGR2Afterwards
15min, 30min and 60min and small animal position emission tomography (PET) imaging figure (D) with the excessive cold closed control group 30min of peptide, arrow generation
Table tumor locus (anterior view, front view)；
Fig. 7 is99mTc-HP-2PisoDGR2In integrin alpha5β1Positive U87MG human glioma tumor model 0.5,1 and 2
Bio distribution result (A) and control group enclosed experiment 0.5h bio distribution result (B), and68Ga-2PisoDGR2Whole
Close element α5β1The bio distribution result (C) and control group of positive U87MG human glioma tumor model 30min and 60min are closed real
Test the bio distribution result (D) of 30min.
Specific embodiment
Material employed in the embodiment of the present invention: (NHS, N- hydroxysuccinimidyl acyl are sub- by N-hydroxysuccinimide
Amine), succinic acid (succinic acid), disodium succinate hexahydrate (disodium succinate), N, N-
Dimethylform amide (DMF, n,N-Dimethylformamide), tricine (trihydroxy methyl glycine) are purchased from the U.S.
Sigma-Aldrich company.Trisodium triphenylphosphine-3,3', 3 "-trisulfonate (TPPTS, three
Phenylphosphine sodium trisulfonate), N, N-Diisopropylethylamine (DIEA, N- ethyl diisopropylamine) are purchased from J&K lark prestige.
1- (3-Dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC.HCl, 1- (3- diformazan
Aminopropyl) -3- ethyl-carbodiimide hydrochloride)) it is purchased from TCI Tokyo Chemical Industry Co., Ltd, Japan.HYNIC-NHS (connection
Hydrazine niacinamide) it is purchased from U.S. Noca-biochem company.PEG4- c (phg-isoDGRk) commission is uncommon to apply biotechnology (Shanghai)
Co., Ltd (U.S. is uncommon to apply biological (CSBio) overseas first branch company) order synthesis.Boc-Glu (NHS)-NHS commission is lucky
You synthesize biochemical 's order.
One kind being based on 2PisoDGR2The radiopharmaceutical of polypeptide, including isoDGR cyclic peptide dimer and radionuclide, institute
Stating isoDGR cyclic peptide dimer is to be connected with bridging agent PEG for two4Ring isoRGD polypeptide monomer dimerization and synthesize,
IsoDGR cyclic peptide dimeric structure is as shown in Figure 1, the sequence of the ring isoRGD polypeptide monomer is different aspartic-glycine-
Arginine；The radionuclide marks the isoDGR cyclic peptide dimer by chelating agent.The chelating agent is HYNIC,
Any one in DOTA, NOTA；The radionuclide is68Ga or99mTc。
Pharmacokinetics decorating molecule company can also be connected between the isoDGR cyclic peptide dimer and the chelating agent
Agent is connect, the pharmacokinetics decorating molecule bridging agent is PEGn, wherein n=1~9.
The radiopharmaceutical is colourless transparent liquid injection.
Embodiment 1
99mTc-HYNIC-PEG4-E[PEG4-c(phg-isoDGRk)]2Preparation:
1)E[PEG4-c(phg-isoDGRk)]2Preparation
PEG will be connected with4The ring isoDGR polypeptide monomer of bridging agent, i.e. PEG4- c (phg-isoDGRk) is dissolved in 1mL DMF；
The glutamic acid Acibenzolar of boc-protected N- hydroxysuccinimide activation, i.e. Boc-Glu (NHS)-NHS is added；It is adjusted with DIEA
PH is adjusted to 8.0-9.0, is stirred overnight at room temperature；Reaction solution separates pure through YMC-Pack ODS-A C18 semi-preparative column HPLC method
Change, collect the fraction of target product, merge collection liquid and is lyophilized；Product is obtained to be confirmed as by MALDI-TOF-MS mass spectral analysis
It is expected that product Boc-E [PEG4-c(phg-isoDGRk)]2；By Boc-E [PEG4-c(phg-isoDGRk)]21mL TFA is added,
Outstanding to boil off except TFA, residue 4mL pure water is dissolved, is lyophilized after filtering；It is true by MALDI-TOF-MS mass spectral analysis to obtain product
Think that expected product is E [PEG4-c(phg-isoDGRk)]2, i.e., the described isoDGR cyclic peptide dimer.
2)HYNIC-PEG4The preparation of-NHS
By amino-PEG4Propionic acid is dissolved in 1mL pure water, adjusts pH to 8.0-9.0 with NaOH；HYNIC-NHS is dissolved in
In 1mL DMF, the amino-PEG for regulating pH is added4Propionic acid aqueous solution；It is stirred overnight at room temperature；Reaction solution is through YMC-Pack
ODS-A C18 semi-preparative column HPLC method isolates and purifies, and collects the fraction of target product, merges collection liquid and is lyophilized；It is produced
Object is confirmed as expected product HYNIC-PEG by MALDI-TOF-MS mass spectral analysis4-COOH；By HYNIC-PEG4- COOH is dissolved in
In 1mL DMF, EDC and NHS is added, is stirred overnight at room temperature；Reaction solution is through the YMC-Pack ODS-A C18 semi-preparative column side HPLC
Method isolates and purifies, and collects the fraction of target product, merges collection liquid and is lyophilized；Product is obtained by MALDI-TOF-MS mass spectrum point
Expected product HYNIC-PEG is confirmed as in analysis4-NHS。
3)HYNIC-PEG4-E[PEG4-c(phg-isoDGRk)]2Preparation
By E [PEG4-c(phg-isoDGRk)]2And HYNIC-PEG4- NHS is dissolved in DMF, adjusts pH to 8.0- with DIEA
9.0, it is stirred overnight at room temperature；Product is isolated and purified through YMC-Pack ODS-A C18 semi-preparative column HPLC method, is collected target and is evaporated
Point, merge collection liquid and is lyophilized；Product is confirmed as expected product HYNIC-PEG by MALDI-TOF-MS mass spectral analysis4-E
[PEG4-c(phg-isoDGRk)]2。
4)99mTc-HYNIC-PEG4-E[PEG4-c(phg-isoDGRk)]2Preparation
Configuration contains TPPTS 5.0mg, tricine 6.5mg, disodium succinate 38.5mg, succinic acid 12.7mg and 30 μ g
HYNIC-PEG4-E[PEG4-c(phg-isoDGRk)]2200 μ L of mixed liquor, the Na of 0.5-1.0mL is added99mTcO4Solution,
100 DEG C of heating water bath cillin bottles react 15-20 minutes, 5 minutes cooling to room temperature after reaction, and 2PisoDGR is made2It is more
Peptide99mTc radiopharmaceutical.
Embodiment 2
99mTc-HYNIC-PEG4-E[PEG4-c(phg-isoDGRk)]2Preparation, on the basis of embodiment 1 further
Optimization.
1)Boc-E[PEG4-c(phg-isoDGRk)]2Preparation
Weigh PEG4- c (phg-isoDGRk) polypeptide 16.6mg (19.8 μm of ol) is dissolved in 1.0mL anhydrous DMF；It weighs simultaneously
Boc-Glu (NHS)-NHS Acibenzolar 4.30mg (10.0 μm of ol) sufficiently dissolution is added in polypeptide solution to mix.It is eventually adding
20.0 μ L DIEA are mixed, and reaction solution is stayed overnight as concussion reaction under room temperature (25 DEG C).Product is carried out by HPLC method 2
Separation and purification collect retention time in the expection product frac of 17.2min.Merge collection liquid freeze-drying, obtains white powder
9.4mg, yield 50%, HPLC examine product purity to be greater than 98%.Product carries out molecular weight through MALDI-TOF-MS mass spectrum
Measurement, mass spectral results are m/z=1885.1 ([M+H]+), with 1885.0 ([C of theoretical molecular weight82H125N21O30]+) be consistent.
2)E[PEG4-c(phg-isoDGRk)]2Preparation
Weigh Boc-E [PEG4-c(phg-isoDGRk)]2Polypeptide 9.4mg, (5.0 μm of ol) are dissolved in 1.0mL TFA.It will be anti-
Answer liquid as concussion reaction 5min under room temperature (25 DEG C).By TFA with being dried with nitrogen, 4mL pure water is added and dissolves residue.After filtering
Filter liquor is lyophilized, white powder 8.9mg, yield 98% are obtained, HPLC method 1 examines product purity to be greater than 98%.Product
The measurement of molecular weight is carried out through MALDI-TOF-MS mass spectrum, mass spectral results are m/z=1785.1 ([M+H]+), with theoretical molecular weight
1784.9([C77H117N21O28]+) be consistent.
3)HYNIC-PEG4The preparation of-COOH
Weigh HYNIC-NHS 45.2mg (100 μm of ol) and amino-PEG4Propionic acid 27.4mg (100 μm of ol) is dissolved in 2.0mL
DMF-H2In O (v:v=1:1).PH to 9.0 is adjusted with 2.0M NaOH, concussion reaction is stayed overnight under room temperature (25 DEG C).Product is led to
The separation and purification of the progress of HPLC method 1 are crossed, collects retention time in the expection product frac of 13.7min.Merge collection liquid to freeze
It is dry, white powder 35.0mg, yield 63% are obtained, HPLC examines product purity to be greater than 98%.Product is through MALDI-TOF-MS
Mass spectrum carries out the measurement of molecular weight, and mass spectral results are m/z=569.2 ([M+H]+), with theoretical molecular weight 568.6
([C24H32N4O10S]+) be consistent.
4)HYNIC-PEG4The preparation of-NHS
Weigh HYNIC-PEG4- COOH 33.0mg (58 μm of ol), EDC.HCL 11.7mg (61 μm of ol) and NHS 7.1mg
(61 μm of ol) is dissolved in 1.0mL DMF, and concussion reaction is stayed overnight under room temperature (25 DEG C).Product is carried out by HPLC method 1
Separation and purification collect retention time in the fraction of 17.2min.Merge collection liquid freeze-drying, obtains white powder 35.0mg, yield
Product purity is examined to be greater than 98% for 63%, HPLC.Product carries out the measurement of molecular weight, mass spectrum knot through MALDI-TOF-MS mass spectrum
Fruit is m/z=666.3 ([M+H]+), with 665.2 ([C of theoretical molecular weight28H35N5O12S]+) be consistent.
5)HYNIC-PEG4-E[PEG4-c(phg-isoDGRk)]2Preparation
Weigh E [PEG4-c(phg-isoDGRk)]2Polypeptide 2.0mg (1.1 μm of ol) and HYNIC-PEG4-NHS 2.0mg
(1.5 μm of ol), is dissolved in 1.0mL DMF.10.0 μ L DIEA are added, concussion reaction is stayed overnight under room temperature (25 DEG C).Product is passed through
The separation and purification that HPLC method 1 carries out collect retention time in the fraction of 24.3min.Merge collection liquid freeze-drying, obtains white
Powder 1.6mg, yield 62%, HPLC examine product purity to be greater than 98%.Product carries out molecule through MALDI-TOF-MS mass spectrum
The measurement of amount, mass spectral results are m/z=2336.3 ([M+H]+), with 2335.5 ([C of theoretical molecular weight101H147N25O37S]+) be consistent
It closes.
6)99mTc-HYNIC-PEG4-E[PEG4-c(phg-isoDGRk)]2Preparation
Configuration contains TPPTS 5.0mg, tricine 6.5mg, disodium succinate 38.5mg, succinic acid 12.7mg and 30 μ g
HYNIC-PEG4-E[PEG4-c(phg-isoDGRk)]2200 μ L of mixed liquor, the Na of 0.5-1.0mL is added99mTcO4Solution,
100 DEG C of heating water bath cillin bottles react 20 minutes, 5 minutes cooling to room temperature after reaction, are made99mTc-HYNIC-PEG4-E
[PEG4-c(phg-isoDGRk)]2。
Wherein:
HPLC method 1: YMC-Pack ODS-A C18 half is equipped with using Agilent 1260Infinity HPLC system and is prepared
Column (250mm × 10mm, 5 μm, 12nm), gradient elution are 30 minutes, flow velocity 4mL/min, and wherein mobile phase A is that pure water is gone (to contain
0.05%TFA), B is acetonitrile (containing 0.05%TFA).Elution gradient is set as 90%A and 10%B when starting, 90%A at 5 minutes
And 10%B, 70%A and 30%B at 25 minutes, 90%A and 10%B at 30 minutes.
HPLC method 2: YMC-Pack ODS-A C18 half is equipped with using Agilent 1260Infinity HPLC system and is prepared
Column (250mm × 10mm, 5 μm, 12nm), gradient elution are 30 minutes, flow velocity 4mL/min, and wherein mobile phase A is that pure water is gone (to contain
0.05%TFA), B is acetonitrile (containing 0.05%TFA).Elution gradient is set as 90%A and 10%B when starting, 90%A at 5 minutes
And 10%B, 70%A and 30%B at 15 minutes, 20%A and 80%B at 25 minutes, 90%A and 10%B at 30 minutes.
Embodiment 3
68Ga-NOTA(DOTA)-E[PEG4-c(phg-isoDGRk)]2Preparation
1)E[PEG4-c(phg-isoDGRk)]2Preparation
PEG will be connected with4The ring isoDGR polypeptide monomer of bridging agent, i.e. PEG4- c (phg-isoDGRk) is dissolved in 1mL DMF；
The glutamic acid Acibenzolar of boc-protected N- hydroxysuccinimide activation, i.e. Boc-Glu (NHS)-NHS is added；It is adjusted with DIEA
PH is adjusted to 8.0-9.0, is stirred overnight at room temperature；Reaction solution separates pure through YMC-Pack ODS-A C18 semi-preparative column HPLC method
Change, collect the fraction of target product, merge collection liquid and is lyophilized；Product is obtained to be confirmed as by MALDI-TOF-MS mass spectral analysis
It is expected that product Boc-E [PEG4-c(phg-isoDGRk)]2；By Boc-E [PEG4-c(phg-isoDGRk)]21mL TFA is added,
Outstanding to boil off except TFA, residue 4mL pure water is dissolved, is lyophilized after filtering；It is true by MALDI-TOF-MS mass spectral analysis to obtain product
Think that expected product is E [PEG4-c(phg-isoDGRk)]2, i.e., the described isoDGR cyclic peptide dimer.
2)NOTA(DOTA)-E[PEG4-c(phg-isoDGRk)]2Preparation
By E [PEG4-c(phg-isoDGRk)]2Polypeptide and NOTA (DOTA)-NHS are dissolved in 1.0mL DMF.With DIEA tune
It saves pH to adjust to 8.0-9.0, be stirred overnight at room temperature；Reaction solution is separated through YMC-Pack ODS-A C18 semi-preparative column HPLC method
The fraction of target product is collected in purifying, is merged collection liquid and is lyophilized；Product is obtained to confirm by MALDI-TOF-MS mass spectral analysis
For expected product NOTA (DOTA)-E [PEG4-c(phg-isoDGRk)]2。
3)68Ga-NOTA(DOTA)-E[PEG4-c(phg-isoDGRk)]2Preparation
From germanium-fresh elution of gallium generator68GaCl3, with the NH of 2.5M4OAc adjusts pH to 3.0~4.0.20 μ g are added
NOTA(DOTA)-E[PEG4-c(phg-isoDGRk)]2, 99 DEG C are heated 20 minutes, system 5 minutes cooling to room temperature after reaction
At 2PisoDGR2Polypeptide68Ga radiopharmaceutical.
Embodiment 4
68Ga-NOTA(DOTA)-E[PEG4-c(phg-isoDGRk)]2Preparation, on the basis of embodiment 3 further
Optimization.
1)Boc-E[PEG4-c(phg-isoDGRk)]2Preparation
Weigh PEG4- c (phg-isoDGRk) polypeptide 16.6mg (19.8 μm of ol) is dissolved in 1.0mL anhydrous DMF；It weighs simultaneously
Boc-Glu (NHS)-NHS Acibenzolar 4.30mg (10.0 μm of ol) sufficiently dissolution is added in polypeptide solution to mix.It is eventually adding
20.0 μ L DIEA are mixed, and reaction solution is stayed overnight as concussion reaction under room temperature (25 DEG C).Product is carried out by HPLC method 2
Separation and purification collect retention time in the expection product frac of 17.2min.Merge collection liquid freeze-drying, obtains white powder
9.4mg, yield 50%, HPLC examine product purity to be greater than 98%.Product carries out molecular weight through MALDI-TOF-MS mass spectrum
Measurement, mass spectral results are m/z=1885.1 ([M+H]+), with 1885.0 ([C of theoretical molecular weight82H125N21O30]+) be consistent.
2)E[PEG4-c(phg-isoDGRk)]2Preparation
Weigh Boc-E [PEG4-c(phg-isoDGRk)]2Polypeptide 9.4mg, (5.0 μm of ol) are dissolved in 1.0mL TFA.It will be anti-
Answer liquid as concussion reaction 5min under room temperature (25 DEG C).By TFA with being dried with nitrogen, 4mL pure water is added and dissolves residue.After filtering
Filter liquor is lyophilized, white powder 8.9mg, yield 98% are obtained, HPLC method 1 examines product purity to be greater than 98%.Product
The measurement of molecular weight is carried out through MALDI-TOF-MS mass spectrum, mass spectral results are m/z=1785.1 ([M+H]+), with theoretical molecular weight
1784.9([C77H117N21O28]+) be consistent.
3)NOTA-E[PEG4-c(phg-isoDGRk)]2Preparation
Weigh E [PEG4-c(phg-isoDGRk)]2Polypeptide 2.0mg (1.1 μm of ol) and NOTA-NHS 2.0mg (1.5 μ
Mol), it is dissolved in 1.0mL DMF.10.0 μ L DIEA are added, concussion reaction is stayed overnight under room temperature (25 DEG C).Product is passed through into HPLC
The separation and purification that (method 1) carries out collect retention time in the fraction of 20.4min.Merge collection liquid freeze-drying, obtains white powder
Last 1.2mg, yield 60%, HPLC examine product purity to be greater than 98%.Product carries out molecular weight through MALDI-TOF-MS mass spectrum
Measurement, mass spectral results be m/z=2235.1 ([M+H]+), with 2234.0 ([C of theoretical molecular weight97H143N25O34S]+) be consistent.
4)68Ga-NOTA-E[PEG4-c(phg-isoDGRk)]2Preparation
Using the HCl of 0.05M, mono- fraction of every 1mL is eluted from germanium-gallium generator68GaCl3, 5mL is eluted altogether.Choose the
1~2mL's68GaCl3, with the NH of 2.5M4OAc adjusts pH to 3.5.20 μ g NOTA-E [PEG are added4-c(phg-isoDGRk)]2, 99
DEG C heating 20 minutes, natural cooling 5min to obtain the final product.Marker is unfolded with Agilent ITLC-SG paper slip, and solvent is
0.1M EDTA-2Na is detected after expansion with radioactivity ITLC.Marker is located at origin, is not marked on polypeptide68Ga
In solvent front.
HPLC method 1: YMC-Pack ODS-A C18 half is equipped with using Agilent 1260Infinity HPLC system and is prepared
Column (250mm × 10mm, 5 μm, 12nm), gradient elution are 30 minutes, flow velocity 4mL/min, and wherein mobile phase A is that pure water is gone (to contain
0.05%TFA), B is acetonitrile (containing 0.05%TFA).Elution gradient is set as 90%A and 10%B when starting, 90%A at 5 minutes
And 10%B, 70%A and 30%B at 25 minutes, 90%A and 10%B at 30 minutes.
HPLC method 2: YMC-Pack ODS-A C18 half is equipped with using Agilent 1260Infinity HPLC system and is prepared
Column (250mm × 10mm, 5 μm, 12nm), gradient elution are 30 minutes, flow velocity 4mL/min, and wherein mobile phase A is that pure water is gone (to contain
0.05%TFA), B is acetonitrile (containing 0.05%TFA).Elution gradient is set as 90%A and 10%B when starting, 90%A at 5 minutes
And 10%B, 70%A and 30%B at 15 minutes, 20%A and 80%B at 25 minutes, 90%A and 10%B at 30 minutes.
HPLC method 3: radioactivity HPLC Quality Control uses Agilent 1260Infinity HPLC system, is furnished with Raytest
Gabi radioactive detector and Agilent-35900E digital analog converter and Agilent Zorbax SB-C18 analytical column
(4.6mm × 250mm, 5 μm), gradient elution are 30 minutes, flow velocity 1mL/min, and wherein mobile phase A is to go pure water (containing 0.05%
TFA), B is acetonitrile (containing 0.05%TFA).Elution gradient is set as 90%A and 10%B when starting, 90%A and 10% at 5 minutes
B, 60%A and 40%B at 25 minutes, 90%A and 10%B at 30 minutes.
The verifying of product effect:
By isoDGR cyclic peptide dimer, that is, E [PEG4-c(phg-isoDGRk)]2With integrin alpha5β1Binding affinity measurement
Integrin alpha is expressed using height5β1U87MG human glioma cell, use125I-c (y-isoRGD-k) is used as integrin alpha5
β1The radioactive ligand of receptor-specific measures E [PEG using competion experiment4-c(phg-isoDGRk)]2IC50Value (half
Inhibition concentration), and isoDGR monomer c (phg-isoDGRk) is set as control.Experimental result shows, E [PEG4-c(phg-
isoDGRk)]2It is competed with c (phg-isoDGRk)125The IC of I-c (y-isoRGD-k) and U87MG cell combination50Value is respectively
13.7 ± 0.56nM and 92.9 ± 5.5nM.(as shown in Figure 3) shows E [PEG4-c(phg-isoDGRk)]2With Integrin α_5 β1
Affinity significantly better than transformation before isoDGR polypeptide.Fig. 3 shows the isoDGR dimer and isoDGR monomer of mtc labeled
Fixed double chamber bed curve in blood.Fast distribution half-life of the isoDGR dimer of mtc labeled in fixed double chamber bed be
1.56min, slow distribution half-life are 24.77min；Fast distribution half-life of the isoDGR monomer of mtc labeled in fixed double chamber bed be
1.42min, slow distribution half-life are 13.79min.The result shows that the clearance rate of the isoDGR dimer of mtc labeled in blood
It is significantly slower than isoDGR monomer, pharmacokinetic property is highly suitable for as Imaging probe.
Fig. 4 is shown99mTc-HP-2PisoDGR2It clearly can image to high contrast U87-MG human glioma tumour
Model, for image results other than kidney has higher intake, other internal organs backgrounds are lower.Receptor blockade tests image results and shows tumour
Intake can be effectively suppressed, and illustrate that tumor imaging is the intake of receptor-specific.Fig. 5 is shown99mTc-HP-2PisoDGR2It can be clear
Image to clear ground high contrast the primary pancreatic tumour model KPC mouse of mouse, it is in vitro analysis shows99mTc-HP-2PisoDGR exists
Normal tissue and organ intake in abdominal cavity is lower, especially very low in the probe intake of Normal Pancreas.And primary pancreatic tumour
With significantly higher tumor uptake, it was demonstrated that the probe has the application potential for imaging primary pancreatic tumour.Fig. 6 is proved
?68Ga-2PisoDGR2U87-MG human glioma tumor model clearly can be imaged to high contrast, image results are in addition to kidney
Have outside higher intake, other internal organs backgrounds are lower.Receptor blockade experiment image results show that tumor uptake can be effectively suppressed, and say
Bright tumor imaging is the intake of receptor-specific.
Fig. 7 is shown99mTc-HP-2PisoDGR2With68Ga-2PisoDGR2Biology in human glioma tumor bearing nude mice
Distribution: being randomly divided into several groups for BALB/c lotus U87MG human glioma nude mice, and every group 4.Each group experiment nude mice passes through respectively
100 μ L of tail vein injection (~74kBq) is different99mTc-HP-2PisoDGR2Or68Ga-2PisoDGR2IsoDGR dimer
Polypeptide extremely tests nude mice by component other places in 15 minutes, 30 minutes, 60 minutes, 120 minutes and 240 minutes after injection, take blood and
Main organs weigh and measure radiocounting, calculate per gram of tissue percentage injection dose rate (%ID/g) after decay correction.
Result verification image results simultaneously show distribution of the probe in each histoorgan.
In conclusion being based on 2PisoDGR2Polypeptide radiopharmaceutical is used for integrin alphavβ6With integrin alpha5β1It is double it is positive, with
And integrin alphavβ6Or integrin alpha5β1The localization diagnosis of single positive tumor patient, has the potentiality for imaging primary cancer of pancreas.
Claims (10)
1. one kind is based on 2PisoDGR2The radiopharmaceutical of polypeptide, it is characterised in that: including isoDGR cyclic peptide dimer and radiation
Property nucleic, the isoDGR cyclic peptide dimer is to be connected with bridging agent PEG for two4Ring isoRGD polypeptide monomer dimerization and
Synthesis, the sequence of the ring isoRGD polypeptide monomer is different aspartic-glycine-arginine；The radionuclide is logical
It crosses chelating agent and marks the isoDGR cyclic peptide dimer.
2. radiopharmaceutical according to claim 1, it is characterised in that: the chelating agent is HYNIC, in DOTA, NOTA
Any one；The radionuclide is68Ga or99mTc。
3. radiopharmaceutical according to claim 1, it is characterised in that: the radionuclide is68Ga, the chelating agent
For DOTA or NOTA.
4. radiopharmaceutical according to claim 1, it is characterised in that: the isoDGR cyclic peptide dimer and the chelating
Pharmacokinetics decorating molecule bridging agent is also connected between agent, the pharmacokinetics decorating molecule bridging agent is PEGn,
Middle n=1 ~ 9.
5. radiopharmaceutical according to claim 4, it is characterised in that: the radionuclide is99mTc, the chelating
Agent is HYNIC.
6. radiopharmaceutical according to claim 5, it is characterised in that: the pharmacokinetics decorating molecule bridging agent is
PEG4。
7. radiopharmaceutical according to claim 1, it is characterised in that: the radiopharmaceutical is colourless transparent liquid needle
Agent.
8. 2PisoDGR described in claim 12The preparation method of polypeptide radiopharmaceutical, it is characterised in that: wherein isoDGR
Cyclic peptide dimer the preparation method comprises the following steps:
PEG will be connected with4The ring isoDGR polypeptide monomer of bridging agent is dissolved in DMF；Boc-protected N- hydroxysuccinimide is added
The glutamic acid Acibenzolar of activation；PH is adjusted with DIEA to adjust to 8.0-9.0；Reaction solution is isolated and purified through HPLC method, collects mesh
The fraction of product is marked, collection liquid is merged and is lyophilized；TFA is added in the product of acquisition, and outstanding to boil off except TFA, residue is dissolved with pure water,
Freeze-drying obtains the isoDGR cyclic peptide dimer after filtering.
9. preparation method according to claim 8, it is characterised in that: including following preparation step:
a. HYNIC-PEGnThe preparation of-NHS:
By amino-PEGnOrganic acid is dissolved in pure water, adjusts pH to 8.0-9.0；HYNIC-NHS is dissolved in DMF, is added and adjusts
Amino-the PEG of good pH valuenAqueous solutions of organic acids；It is stirred at room temperature；Reaction solution is isolated and purified through HPLC method, collects target product
Fraction, merge collection liquid simultaneously be lyophilized, obtain HYNIC-PEGn-COOH；By HYNIC-PEGn- COOH is dissolved in DMF, and EDC is added
And NHS, it is stirred at room temperature；Reaction solution is isolated and purified through HPLC method, collects the fraction of target product, is merged collection liquid and is lyophilized and obtains
Obtain HYNIC-PEGn-NHS；
B.HYNIC-PEGn-E[PEG4-c(phg-isoDGRk)]2Preparation:
The isoDGR cyclic peptide dimer is expressed as E [PEG4-c(phg-isoDGRk)]2, by isoDGR cyclic peptide dimer and
HYNIC-PEGn- NHS is dissolved in DMF, is adjusted pH to 8.0-9.0 with DIEA, is stirred at room temperature；Product separates pure through HPLC method
Change, collect target fraction, merges collection liquid and freeze-drying obtains HYNIC-PEGn-E[PEG4-c(phg-isoDGRk)]2；
c. 99mTc -HYNIC-PEGn-E[PEG4-c(phg-isoDGRk)]2Preparation:
Configuration contains TPPTS, tricine, disodium succinate, succinic acid and HYNIC-PEGn-E[PEG4-c(phg-
isoDGRk)]2Mixed liquor, be added Na99mTcO4Solution, 100 DEG C heating in water bath for reaction 15-20 minutes, to room after reaction
Temperature is cooling, is made99mTc -HYNIC-PEGn-E[PEG4-c(phg-isoDGRk)]2, as it is based on 2PisoDGR2Polypeptide is put
Penetrating property drug.
10. preparation method according to claim 8, it is characterised in that: including following preparation step:
a. NOTA(DOTA)-E[PEG4-c(phg-isoDGRk)]2Preparation:
The isoDGR cyclic peptide dimer is expressed as E [PEG4-c(phg-isoDGRk)]2, by the isoDGR cyclic peptide dimer and
NOTA-NHS or DOTA-NHS is dissolved in 1.0 mL DMF, is adjusted pH with DIEA and is adjusted to 8.0-9.0, is stirred at room temperature；Reaction solution
It is isolated and purified through HPLC method, collects the fraction of target product, merge collection liquid and be lyophilized, obtain product NOTA (DOTA)-E
[PEG4-c(phg-isoDGRk)]2；
b. 68Ga-NOTA(DOTA)-E[PEG4-c(phg-isoDGRk)]2Preparation:
From germanium-fresh elution of gallium generator68GaCl3, use NH4OAc adjusts pH to 3.0 ~ 4.0, and NOTA (DOTA)-E is added
[PEG4-c(phg-isoDGRk)]2, 99 DEG C are heated 19 ~ 21 minutes, and it is cooling to room temperature after reaction, it is made68Ga-NOTA
(DOTA)-E[PEG4-c(phg-isoDGRk)]2, as it is based on 2PisoDGR2The radiopharmaceutical of polypeptide.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110746492A (en) * | 2019-11-20 | 2020-02-04 | 南京恒远科技开发有限公司 | Preparation process of hydrazine-linked nicotinamide polyethylene glycol bicyclic pentapeptide |
| WO2023143502A1 (en) * | 2022-01-29 | 2023-08-03 | 中国科学院生物物理研究所 | FAP-α SPECIFIC RADIOPHARMACEUTICAL AND APPLICATION THEREOF |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107325169A (en) * | 2017-07-02 | 2017-11-07 | 南京市第医院 | It is a kind of68Class Exenatide compound of Ga marks and its preparation method and application |
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2019
- 2019-05-08 CN CN201910379030.0A patent/CN110101880A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107325169A (en) * | 2017-07-02 | 2017-11-07 | 南京市第医院 | It is a kind of68Class Exenatide compound of Ga marks and its preparation method and application |
Non-Patent Citations (1)
| Title |
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| HANNAN GAO ET AL: "Improved in Vivo Targeting Capability and Pharmacokinetics of 99mTc-Labeled isoDGR by Dimerization and Albumin-Binding for Glioma Imaging", 《BIOCONJUGATE CHEM.》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110746492A (en) * | 2019-11-20 | 2020-02-04 | 南京恒远科技开发有限公司 | Preparation process of hydrazine-linked nicotinamide polyethylene glycol bicyclic pentapeptide |
| WO2023143502A1 (en) * | 2022-01-29 | 2023-08-03 | 中国科学院生物物理研究所 | FAP-α SPECIFIC RADIOPHARMACEUTICAL AND APPLICATION THEREOF |
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